PROJECT SUMMARY While neurodegenerative disorders have different proximal causes and affect different neuronal types, nearly all neurodegenerative disorders, including Alzheimer?s disease, Parkinson?s disease, and amyotrophic lateral sclerosis, have one pathological feature in common?an abnormal accumulation of DNA damage in neurons. When improperly repaired, DNA damage can generate somatic mutations that riddle the genome and promote neuronal dysfunction and loss. These mutagenic processes are operational in the aging brain and are accelerated in Alzheimer?s disease and other neurodegenerative diseases. Many somatic mutations in neurons originate from DNA damage incurred during active transcription. Topoisomerase 1 (TOP1) relieves torsional stress in DNA during transcription and dysfunction of TOP1 is associated with several human neurodegenerative disorders. However, it is unknown if certain neuronal subtypes are more vulnerable to TOP1 dysfunction or if pathological effects associated with transcription-driven DNA damage can be minimized therapeutically. Recently, we found that acute inhibition or acute deletion of Top1 caused biased downregulation of extremely long genes in cultured cortical neurons. Many long genes are associated with synaptic transmission. To evaluate how chronic loss of Top1 affects neuronal functions, we conditionally deleted (cKO) Top1 in postmitotic excitatory neurons of the mouse cerebral cortex. In preliminary studies, we found that these mice showed signs of DNA damage in excitatory neurons, neuroinflammation, and neuron loss beginning around postnatal day 7. These hallmarks of neurodegeneration were accompanied by behavioral decline and death by postnatal day 24. Deletion of p53, a core component of the classic DNA damage response pathway, partially rescued cortical neuron degeneration but did not rescue behavioral decline or death. Single cell (sc)DNA-seq experiments suggest that Top1 cKO neurons accumulate an abnormal number of somatic mutations prior to their demise. Based on these and other data, we centrally hypothesize that deletion of Top1 in postmitotic neurons causes neurodegeneration by impairing transcriptional processes and promoting the accumulation of damaging somatic mutations in neurons. To test this central hypothesis, we will: 1) resolve the timing of when DNA damage occurs relative to upper and lower layer cortical neuron loss, 2) use scRNA-seq and scDNA-seq to identify molecular and genomic changes that precede neurodegeneration in Top1 cKO cerebral cortex. Lastly, 3) we will use genetic and pharmacological approaches to probe the mechanism of Top1 cKO-mediated neurodegeneration. The trajectory of neurodegeneration is similar in culture and in mice. Cortical neuron loss in culture was fully rescued by re-expressing TOP1 and partially rescued by treating with a neuroprotective drug. Unlike other animal models of neurodegeneration, the rapid (< 25 day) and predictable time course of decline and death make Top1 cKO mice ideal for testing candidate neuroprotective compounds in vivo.